The present invention relates to a multi-colored electrophoresis pattern reading system and, more particularly, to a multi-colored electrophoresis pattern reading system appropriate for the comparison of a plurality of patterns of electrophoresis by labelling each of samples with fluorescent pigments having different fluorescent wavelengths, subjecting the samples to electrophoresis simultaneously, and reading the resulting patterns of electrophoresis.
Generally, electrophoresis analysis method using samples labeled with a radioactive isotope has been employed for analyzing the sequences of various genes, including diagnosis of diseases deriving from genes, the structures of proteins such as amino acids, etc. The electrophoresis analysis method is a method for analyzing samples by labelling or replacing fragments of a sample with or by a radioactive isotope, subjecting the fragments of the sample to electrophoresis with a gel, and analyzing a pattern of distribution of the fragments of the sample developed by means of electrophoresis.
Description will now be made of the diagnosis of hereditary diseases as an example for reading and analyzing patterns of electrophoresis. The human gene DNA consists of pairs of bases of approximately 3.times.10.sup.9 and the sequences of the bases are generally constant among the human beings, although there is a deviation in the sequences of the bases among individuals. This deviation is called a polymorphism of the DNA. The polymorphism of the DNA is seen in the non-hereditary region as well as in the hereditary region and the polymorphism of the DNA appears in many occasions as a polymorphism of proteins that is a phenotype of the polymorphism of the DNA. A variety of variations as seen among the human beings, such as the blood types, histocompatible antigens, the difference of skin and hair colors among peoples, etc. are based on the polymorphism of the DNA. The polymorphism of the DNA has been created on the basis of variations that have been accumulated in the DNA of the genocytes of the human groups up to the present time from the time when the human beings were developed as an individual biological species in the course of the evolution of the human beings. When such a variation exists in the site that has the function of significance in terms of the existence as an individual person and when a nosogenic phenotype resulting from the variation occurs as a pathologic state, the pathologic state is called a hereditary disease. It is said that there are currently more than 3,000 kinds of hereditary diseases in the human group.
The nosogenesis of the hereditary disease is an abnormality appeared on the DNA sequence. However, it is recognized for the first time as a disease in several stages ranging from a DNA through a mRNA and proteins to pathogenic phenotype. The diagnosis as a disease is conducted usually in the last stage and the diagnosis can be implemented at the DNA level or at the protein level if the disease would occur simply in the course of the several stages as described hereinabove.
The basic technique for the diagnosis of a DNA is called Southern plotting that basically consists of six steps:
Step 1: Extraction of a DNA as a sample;
Step 2 Fragmentation of the DNA with restricting enzymes;
Step 3: Fractionation of DNA fragments by molecular weights through gel electrophoresis;
Step 4: Migration of DNA fractions to filter;
Step 5: Hybridization of the DNA fractions with a probe DNA (obtained by labelling a DNA having a hemeomorphous sequence of the gene to be detected); and
Step 6: Detection of the hybrid by autoradiography.
For the diagnosis of the hereditary diseases, the DNA extracted from any organ is employed and the sample required for that purpose is usually the peripheral blood of the order of several milliliters. The DNA is extracted from the leukocytes separated from the peripheral blood as the sample. Approximately five days are usually required from step 1 to step 6. In diagnosing the hereditary diseases, the pattern of a fraction from a person tested is compared with the pattern of a fraction from a normal person. The person tested is decided as normal when the pattern of the fraction from the person tested is determined to be identical to the pattern of the fraction from the normal person.
Recently, attempts have been made to conduct tests by using a probe DNA labelled with a fluorescent pigment, in place of a radioactive isotope, to excite the fluorescent pigment and to read the pattern of electrophoresis, from the point of view of safety and other environmental problems. However, highly sophisticated optical and signal processing techniques capable of detecting a faint magnitude of light are required to give a signal-to-noise ratio as equivalent as the radioactive isotope is employed, because the quantity of the sample required for diagnosis of the hereditary disease and determination of the sequence of bases is of the order of approximately 10.sup.-15 mole.
Japanese Patent Laid-open Publication (kokai) No. 62,843/1986 (corresponding to U.S. Pat. No. 4,675,059) discloses an electrophoresis apparatus of a fluorescence detecting type, capable of detecting a minute quantity of a sample labelled with a fluorescent pigment.
A description will now be made of such an electrophoresis apparatus based on fluorescence detection method.
FIG. 18 is a perspective view showing an outlook of a conventional electrophoresis apparatus of a fluorescent type. The electrophoresis apparatus comprises a combined electrophoresis and instrumentation unit 51 for implementing electrophoresis of a sample and measuring the distribution of fluorescence, a data processor 52 for performing data processing on the basis of measured data, and a cable 53 connecting the combined electrophoresis and instrumentation unit 51 to the data processor 52. The electrophoresis and instrumentation unit 51 has a door 51a through which a gel serving as a base for performing electrophoresis for DNA fragments and a predetermined quantity of a sample (DNA fragments) for electrophoresis are poured into the electrophoresis and instrumentation unit 51. As the door 51a is closed and a switch for starting electrophoresis on an operation display panel 51b is pressed to start electrophoresis. As the electrophoresis has been started, a monitor of the operation display panel 51b of the electrophoresis and instrumentation unit 51 displays an operational state. The data measured by the electrophoresis and instrumentation unit 51 is transmitted to the data processor 52 in which the data is processed on the basis of a predetermined program stored in advance. The data processor 52 comprises a computer body 54, a keyboard 55 for entering an instruction from the operator, a display unit 56 for displaying the processing state and results, and a printer 57 for recording the data-processed results.
FIG. 19 is a block diagram showing the configuration of the inside of the electrophoresis and instrumentation unit. As shown in FIG. 19, an overall configuration of the combined electrophoresis and instrumentation unit 51 (FIG. 18) comprises an electrophoresis unit section 63 and a signal processor unit section 64. The electrophoresis unit section 63 comprises an electrophoresis section 5 for implementing electrophoresis, a first electrode 2a and a second electrode 2b each for applying voltage to the electrophoresis section 5, a support plate 3 for supporting the electrophoresis section 5 as well as the first and second electrodes 2a and 2b, a power source unit 4 for applying voltage to the electrophoresis section 5, a light source 11 for emitting light for exciting a fluorescent substance, an optical fiber 12 for leading the light from the light source 11, a light collector 14 of an optic system for condensing and collecting fluorescence 13 generated by the fluorescent substance, an optical filter 15 for selectively passing the light having a particular wavelength therethrough, and an optical sensor 16 for converting the condensed light into electric signals. The signal processor unit section 64 comprises an amplifier 17 for amplifying the electric signals from the optical sensor 16, an analog-digital converting circuit 18 for converting analog signals of the electric signals into digital data, a signal processing section 19 for pre-processing the digital data, for example, by performing addition average processing or the like, an interface 20 for implementing interface processing for feeding the pre-processed data to an external data processor, and a control circuit 10 for implementing overall control of the electrophoresis unit section and the signal processing system. The digital signal OUT generated from the signal processor unit section 64 is transmitted to the data processing unit 52 (FIG. 18), thereby implementing the data processing such as analysis processing and so on.
A description will now be made of the operation of the electrophoresis apparatus with reference to FIGS. 18 and 19.
After the door 51a of the electrophoresis and instrumentation unit 51 is opened, a gel is poured into the electrophoresis section 5 disposed within the combined electrophoresis and instrumentation unit 51 and thereafter a sample of DNA fragments labelled with a fluorescent substance is poured thereinto. As a switch of the instrument panel 51b is pressed to give an instruction to start electrophoresis, then voltage is applied from the first and second electrodes 2a and 2b of the power source unit 4 to the electrophoresis section 5, thereby starting the electrophoresis. The electrophoresis allows the sample labelled with the fluorescent substance to migrate, for example, in lanes 71, 72, 73 and 74, as shown in the schematic representation 70 of FIG. 22, gathering the molecules having the same molecular weights together forming bands 66. The molecules having lower molecular weights migrate faster than those having higher molecular weights so that the former migrates in a distance longer than the latter within the same time unit. The bands 66 are detected in a manner as shown in FIG. 20a by leading light from the light source through the optical fiber 12 to a light path 61 and irradiating the gel on the light path 61 with the light, exciting the labelled fluorescent substance concentrated on the bands 66 in the gel to generate fluorescence 13, and detecting the fluorescence 13. The fluorescence 13 generated contains the fluorescent substance in the concentration as extremely low as approximately 10.sup.-16 mole per band, although the quantity of fluorescence may depend upon an extinction coefficient of the fluorescent substance used, quantum efficiency thereof, intensity of exciting light, etc. For instance, fluorescein isothiocyanate has a peak of the wavelength of excitation at 490 nm, a peak of its fluorescent wavelength of 520 nm, an extinction coefficient of 7.times.104 mole.sup.-1. mole.sup.-1, and a quantum efficiency of approximately 0.65. If fluorescein isothiocyanate employed exists in the concentration of 10.sup.-16 mole per band, the fluorescence generated contains photons of the order as low as 10.sup.10 /S, when calculated by postulating the use of argon ion laser of a wavelength of 488 nm at output of 1 mW as a fluorescent substance, although it may vary to some extent with the thickness of the gel or the like. Hence, an extremely faint magnitude of fluorescence is required to be detected.
Referring to a front view as shown in FIG. 20a and to a longitudinally sectional view as shown in FIG. 20b, the electrophoresis section 5 comprises a gel member 5a composed of polyacrylamide or the like and gel supporting members 5b and 5c, each made of glass for supporting and interposing the gel member 5a from the both sides. A sample of DNA fragments is poured into the gel member 5a of the electrophoresis section 5 from its upper portion and the electrophoresis is carried out by applying voltage for electrophoresis to the first electrode 2a and the second electrode 2b (FIG. 18). While the electrophoresis is being carried out, the fluorescent substance contained in the bands of the pattern of electrophoresis in the gel member 5a along the light path 61 is irradiated with rays of light sent out from the light source, such as laser light, which pass through the optical fiber 12 onto the light path 61 of the gel member 5e. This allows the fluorescent substance present on the light path 61 to be excited to emit fluorescence 13 that is led to a light collector 14 of optics consisting of a combination of lenses and then selected by the optical filter 15 after having been condensed, followed by conversion into electric signals by means of a one-dimensional optical sensor 16. In order to convert a faint quantity of light into electric signals in an efficient fashion, the light is amplified to 10.sup.4 to 10.sup.5 times with an image intensifier or the like, and the image is converted into electric signals by the optical sensor 16, such as a one-dimensional optical sensor of CCD or the like. The electric signals converted by the optical sensor 16 are then amplified to signals of a desired level by the amplifier 17, and the analog signals are converted into digital signals by the analog-digital converting circuit 18, followed by transmission to the signal processing section 19. The digital signals transmitted from the analog-digital converting circuit 18 are then subjected to signal processing, such as addition average processing, or the like, in order to improve the signal-to-noise ratio (an S/N ratio), and the resulting digital data is transmitted to the data processor unit section 52 through the interface 20.
FIGS. 21a and 21b are schematic representations for describing an example of signals of a pattern indicative of the fluorescent intensity of DNA fragments transmitted from the electrophoresis and instrumentation unit 51. For instance, as shown in FIG. 21a, the fluorescent substance present on the light path 61 is excited upon irradiation of the gel member 5a of the electrophoresis section 5 with the laser light in the course of electrophoresis, thereby emitting fluorescence. The fluorescence is detected at predetermined positions of each lane in the direction of electrophoresis, as indicated by 62, as the time of electrophoresis elapses. In other words, the fluorescence is detected as the bands 66 of each lane pass through the positions of the light path 61, thereby detecting a pattern signal of fluorescence intensity in each of the lanes, as shown in FIG. 21b. As a peak of the fluorescence intensity is given When each of the bands 66 passes through the positions of the light path 61, the pattern signal of the fluorescence intensity as shown in FIG. 21b represents a pattern signal indicating the magnitude of fluorescence intensity of the bands 66 located in the direction of electrophoresis, as indicated by 62. In other words, the pattern signal constitutes a profile wave form proportional to the concentration of fluorescence, and a sequence of the bases of a DNA fragment may be determined by deciding a peak value of the pattern signal.
The computer body 54 of the data processing unit 52 implements data processing for comparing molecular weights and determining a sequence of bases of a DNA fragment on the basis of data of the pattern indicative of fluorescence intensity in response to data of the pattern signals for the fluorescence intensity of the DNA fragments transmitted from the electrophoresis and instrumentation unit 51. The sequence of the bases and so on determined by the data processing is symbolized and then generated, thereby displaying the symbolized data on a display screen by the display unit 56 or printing it out by the printer 57.
The aforesaid embodiment is directed to an example of the apparatus in which the fluorescent pigment is employed for labelling the sample. Japanese Patent Laid-open Publication (kokai) No. 167,649/1989 discloses another example of an apparatus capable of reading a fluorescent pattern of electrophoresis. This apparatus is of such a type as reading a fluorescent pattern of the electrophoresis section as a whole after the end of electrophoresis, unlike the aforesaid electrophoresis apparatus of such a type as reading the distribution of the fluorescent pattern passing through a reading section in the course of electrophoresis.
It is to be noted herein that the gel electrophoresis method employed for the electrophoresis pattern reading apparatuses on the basis of the fluorescence detection method is the same as the gel electrophoresis method which has been employed for the conventional apparatuses in which the sample is labeled with the radioactive isotope. The gel electrophoresis method may cause a warp in the pattern of electrophoresis because a speed of migration of bands may vary with the position of an electrophoresing plate due to irregularities in temperatures within the gel and for other reasons, thereby causing a warp in the pattern of electrophoresis. Hence, for example, when electrophoresis of two kinds of samples or electrophoresis in a two-dimensional way is to be performed with the purpose to compare two kinds of patterns of electrophoresis for the diagnosis of hereditary diseases, the positions of the electrophoresed bands may be deviated between the results of electrophoresis due to the warp and a comparison between the two patterns of electrophoresis may be rendered difficult, even if either of the methods for labelling the samples with the radioactive isotope or with the fluorescent substance would be adopted, as long as the conventional gel electrophoresis method is employed. Further, implementation of the correction of such patterns of electrophoresis by means of data processing is also rendered laborious and complex.
Further, as the electrophoresis and instrumentation unit for implementing electrophoresis and simultaneously measuring the distribution of the fluorescent substance passing through the reading unit adopts two-dimensional electrophoresis, it requires the one-dimensional electrophoresis to be implemented by one device and the two-dimensional electrophoresis to be conducted by another device, so that this operation is laborious and complex.